Cloning, characterization, and modeling of a monoclonal anti‐human transferrin antibody that competes with the transferrin receptor

Orlandini, Maurizio; Santucci, Annalisa; Tramontano, Anna; Neri, Paolo; Oliviero, Salvatore

doi:10.1002/pro.5560030913

Cited by 9 publications

(11 citation statements)

References 40 publications

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“…Taken together, the immunoband shift assays support that α2,3-and α2,8-linked sialic acids were attached to the N-glycans of NCAM, as previously shown [43,44]. The electrophoretic migration of transferrin (94 kDa) from untreated membranes ( Figure 2B) was similar to previous reports [45,46]. Moreover, we observed that the electrophoretic migration of transferrin synthesized in liver was virtually identical with that in brain (results not shown).…”

Section: Neuronal Kv3 Proteins Are Associated With α23-and α28-linksupporting

confidence: 91%

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Cartwright

Schwalbe

2009

Bioscience Reports

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Mammalian brains contain relatively high amounts of common and uncommon sialylated N-glycan structures. Sialic acid linkages were identified for voltage-gated potassium channels, Kv3.1, 3.3, 3.4, 1.1, 1.2 and 1.4, by evaluating their electrophoretic migration patterns in adult rat brain membranes digested with various glycosidases. Additionally, their electrophoretic migration patterns were compared with those of NCAM (neural cell adhesion molecule), transferrin and the Kv3.1 protein heterologously expressed in B35 neuroblastoma cells. Metabolic labelling of the carbohydrates combined with glycosidase digestion reactions were utilized to show that the N-glycan of recombinant Kv3.1 protein was capped with an oligo/poly-sialyl unit. All three brain Kv3 glycoproteins, like NCAM, were terminated with alpha2,3-linked sialyl residues, as well as atypical alpha2,8-linked sialyl residues. Additionally, at least one of their antennae was terminated with an oligo/poly-sialyl unit, similar to recombinant Kv3.1 and NCAM. In contrast, brain Kv1 glycoproteins consisted of sialyl residues with alpha2,8-linkage, as well as sialyl residues linked to internal carbohydrate residues of the carbohydrate chains of the N-glycans. This type of linkage was also supported for Kv3 glycoproteins. To date, such a sialyl linkage has only been identified in gangliosides, not N-linked glycoproteins. We conclude that all six Kv channels (voltage-gated K+ channels) contribute to the alpha2,8-linked sialylated N-glycan pool in mammalian brain and furthermore that their N-glycan structures contain branched sialyl residues. Identification of these novel and unique sialylated N-glycan structures implicate a connection between potassium channel activity and atypical sialylated N-glycans in modulating and fine-tuning the excitable properties of neurons in the nervous system.

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Section: Neuronal Kv3 Proteins Are Associated With α23-and α28-linksupporting

confidence: 91%

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Cartwright

Schwalbe

2009

Bioscience Reports

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“…Analysis of the sequences revealed that the MAbs used different germ line family sequences for V H , V, and J but the same germ line family for J H ( Table 1). Comparison of these heavy-and light-chain rearranged V region sequences to similar sequences found in the GenBank database has shown that the V region rearrangement events encode various immunoglobulin binding specificities ranging from double-stranded DNA to protein epitopes (3,4,22,(26)(27)(28)30).…”

Section: Sequencing Of Mab Variable Domainsmentioning

confidence: 99%

Specificity and Diversity of Antibodies toMycobacterium tuberculosisArabinomannan

Navoa¹,

Laal

Pirofski³

et al. 2003

Clin Vaccine Immunol

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Arabinomannan (AM) is a polysaccharide antigen of the mycobacterial capsule. However, it is uncertain whether AM constitutes an immunologically distinct fraction of Mycobacterium tuberculosis. In this study, we analyzed the repertoire and specificity of antibodies to AM by using AM-binding murine monoclonal antibodies (MAbs) and human serum samples. Murine MAbs were found to be diverse in their specificity to AM and cross-reactivity with other arabinose-containing mycobacterial polysaccharides, with MAb 9d8 binding exclusively to AM. Human antibodies to AM were detected in serum samples from patients with pulmonary tuberculosis (TB), as well as in those from healthy, purified protein derivative-negative controls, with significantly higher titers among patients. The binding of human antibodies to AM was inhibited by MAb 9d8 in three patients with TB but not in controls. MAb 5c11, which recognizes other mycobacterial arabinosecontaining carbohydrates in addition to AM, inhibited the binding of serum samples from 75% of patients and 76% of controls. Analysis of human antibodies with murine MAbs to human V H determinants demonstrated diversity among antibodies to AM with qualitative and quantitative differences compared with antibodies to lipoarabinomannan. In summary, our study suggests that antibodies to AM are diverse and heterogeneous with respect to antigen recognition and V H determinant expression, with human serum samples containing different subsets of antibodies to AM with the specificities of AM-binding murine MAbs. One MAb and a subset of human antibodies bind AM specifically, suggesting that this polysaccharide is antigenically distinct and is expressed in human infection.

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“…The sequences of thousands of antibodies of the IgG class have been determined (Kabat et al, 1991). However, the three-dimensional structures of only a small subset of sequenced antibodies have been determined by x-ray crystallography, but several have been homology-modeled (Anchin et al, 1991;Bassolino-Klimas et al, 1992;Mas et al, 1992;Tanner et al, 1992;Barry et al, 1994;Orlandini et al, 1994;Tenette-Souaille and Smith, 1998;Tenette et al, 1996).…”

Section: Introductionmentioning

confidence: 99%

Modeling the Binding Sites of Anti-Hen Egg White Lysozyme Antibodies HyHEL-8 and HyHEL-26: An Insight into the Molecular Basis of Antibody Cross-Reactivity and Specificity

Srinivasan¹,

Sinha²,

Smith‐Gill

2003

Biophysical Journal

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Three antibodies, HyHEL-8 (HH8), HyHEL-10 (HH10), and HyHEL-26 (HH26) are specific for the same epitope on hen egg white lysozyme (HEL), and share >90% sequence homology. Their affinities vary by several orders of magnitude, and among the three antibodies, HH8 is the most cross-reactive with kinetics of binding that are relatively invariable compared to HH26, which is highly specific and has quite variable kinetics. To investigate structural correlates of these functional variations, the Fv regions of HH8 and HH26 were homology-modeled using the x-ray structure of the well-characterized HH10-HEL complex as template. The binding site of HH26 is most charged, least hydrophobic, and has the greatest number of intramolecular salt bridges, whereas that of HH8 is the least charged, most hydrophobic and has the fewest intramolecular salt bridges. The modeled HH26-HEL structure predicts the recently determined x-ray structure of HH26, (Li et al., 2003, Nat. Struct. Biol. 10:482-488) with a root-mean-square deviation of 1.03 A. It is likely that the binding site of HH26 is rendered rigid by a network of intramolecular salt bridges whereas that of HH8 is flexible due to their absence. HH26 also has the most intermolecular contacts with the antigen whereas HH8 has the least. HH10 has these properties intermediate to HH8 and HH26. The structurally rigid binding site with numerous specific contacts bestows specificity on HH26 whereas the flexible binding site with correspondingly fewer contacts enables HH8 to be cross-reactive. Results suggest that affinity maturation may select for high affinity antibodies with either "lock-and-key" preconfigured binding sites, or "preconfigured flexibility" by modulating combining site flexibility.

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Cloning, characterization, and modeling of a monoclonal anti‐human transferrin antibody that competes with the transferrin receptor

Cited by 9 publications

References 40 publications

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Atypical sialylated N-glycan structures are attached to neuronal voltage-gated potassium channels

Specificity and Diversity of Antibodies toMycobacterium tuberculosisArabinomannan

Modeling the Binding Sites of Anti-Hen Egg White Lysozyme Antibodies HyHEL-8 and HyHEL-26: An Insight into the Molecular Basis of Antibody Cross-Reactivity and Specificity

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